Genetic Analysis of T-cell Differentiation

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

We study the transcriptional control of T cell development and function. T cells are essential for immune responses. Most recognize peptide antigens presented by class I (MHC-I) or class II (MHC-II) classical Major Histocompatibility Complex molecules, and express either of two surface glycoproteins that contribute to antigen recognition: CD4, which binds MHC-II, or CD8, which binds MHC-I. Consistent with such binding properties, MHC I-specific T cells generally are CD4-CD8+ (CD8 T cells), whereas MHC II-specific T cells generally are CD4+CD8- (CD4 T cells). CD4 T cells are essential for life: CD4 T cell deficiency, whether innate or acquired, leads to recurrent or chronic infections and death. CD4 T cell responses have remained an essential area of focus for the laboratory. After infection or immunization (e.g. vaccine), antigen-specific CD4 T cells proliferate and, depending on the local inflammatory context, differentiate into effector subtypes endowed with specific functions. Infections by intra-cellular pathogens (e.g. viruses) result in the generation of Th1 CD4 T cells producing interferon (IFN)-gamma and of follicular helper CD4 T cells (Tfh), which provide help to B cells for antibody maturation. Additionally, long-lived memory CD4 T cells, persisting after pathogen clearance, contribute to durable immunity, together with the B cell response. Accordingly, efficient differentiation of Tfh and memory CD4 T cells are key objectives of vaccination strategies. To gain insight into T cell development and function, a major objective of the laboratory in recent years has been to implement single cell (sc) analyses of gene expression by RNA sequencing (scRNAseq). We recently extended these studies to chromatin accessibility (ATAC sequencing, scATACseq), which provides a broader view of the cell functional potential than analyses of actual gene expression. Our recent and ongoing research has exploited these methods to study CD4 and CD8 T cell responses, including in viral infections (Ciucci et al., 2019; Vacchio et al., 2019; Jaiswal et al., 2021) and in the tumor micro-environment (Magen et al., 2019). A recent study from the laboratory (Chopp et al., 2020) leveraged these approaches to explore intrathymic T cell development. We have focused on the differentiation of cells expressing an alpha-beta T cell antigen receptor (TCR), which comprise not only mainstream CD4 and CD8 lineage T cells, but also multiple lineages involved in immune homeostasis (e.g. regulatory T cell, Treg) or innate-like responses (e.g. natural killer [NK]) T cells). Because of the allelic diversity of MHC molecules, most alpha-beta TCR-expressing thymocytes die by neglect via programmed cell death as they fail to productively interact with intrathymic MHC peptide complexes. Among those that escape this fate, thymocytes of high affinity for intrathymic ligands are targeted for active deletion (negative selection), or for differentiation into agonist-selected fates, including Treg cells. Appropriate deletion of self-reactive cells or their diversion to such regulatory lineages is essential to prevent the emergence of auto-immune disease. In contrast, cells with moderate affinity for self MHC peptide are rescued from cell death (a process called positive selection), and become conventional CD4 and CD8 T cells. Our long-term objectives in these studies are (i) to build developmental trajectories leading CD4- and CD8-expressing (double-positive, DP) thymocyte precursors to these various lineages, (ii) to identify relevant transcriptomic patterns and cis-regulatory elements (from scRNAseq and scATACseq, respectively), and (iii) to infer gene regulatory networks controlling these processes. Although genetics, flow cytometry, and population-based analyses of gene expression have provided important insight into these questions, they are limited by the multiplicity and developmental heterogeneity of alpha-beta lineages, and by their clonotypic diversity (i.e. each developing thymocyte expresses a single TCR specificity). In contrast, single cell analyses of gene expression and chromatin accessibility are ideally suited to overcome these limitations. Our study (Chopp et al., 2020) provided insight into the differentiation of DP thymocytes and their progeny. Unbiased mapping of the transcriptome identified differentiation pathways for conventional CD4 and CD8 T cells: we found that CD4- and CD8-lineage differentiation programs emerged asynchronously, at early and late stages of thymocyte development, respectively. We also identified agonist-selected developmental trajectories, including distinct transcriptomic and chromatin accessibility profiles in cells signaled by MHC-I vs. MHC-II molecules. The integration of scRNAseq and scATACseq demonstrated a strong correspondence between transcriptome and chromatin accessibility and identified chromatin regions with lineage- or stage-specific accessibility, e.g. near the gene encoding the pro-apoptotic protein Bim in agonist-signaled thymocytes. In collaboration with Sridhar Hannenhalli's group at NCI, we computed gene regulatory networks from integrated transcriptomic and epigenomic data; this identified transcription factors putatively associated with differentiation into CD4 or CD8 lineages. In parallel, using chromatin immunoprecipitation of differentiating CD4-lineage thymocytes, we mapped their DNA binding sites for Thpok, a transcription factor required for CD4 T cell differentiation. Last, we demonstrated that the transcriptomic and epigenomic patterns that we identified in mouse thymocytes are largely conserved in scRNAseq and scATACseq data we generated from human thymocytes. Altogether, the comprehensive data sets generated in this study (all publicly available from NCBI repositories) provide new bases to decipher the mechanisms driving alpha-beta T cell development.